Enzymes are biological proteins that speed up chemical reactions by lowering the energy barrier for them to occur more easily. Enzymes demonstrate a high degree of utility due to their speed of reaction, specificity for certain analytes, and ability to be engineered and chemically modified. Enzymes are used in many industries including food processing, detergents and cleaning products, clinical diagnostics, fuel production and decontamination of chemical agents. The major problem associated with the practical utility of enzymes is the inability to sufficiently stabilize their tertiary structure in harsh environmental conditions, such as high temperatures, extreme pH, high salinity and solvents; free enzymes are susceptible to damage and will incur partial or total activity loss in the presence of such conditions. As a result, applications of free enzymes for large scale commercial use, especially for continuous use, are extremely limited. The ability to stabilize enzymes in harsh conditions is an area of immense interest; retention of activity in non optimized environments, such as elevated temperatures, will improve catalytic performance and be beneficial for countless applications.
Various approaches for stabilizing enzymes have been demonstrated from enzyme adsorption and modification to recombinant protein engineering; these methods only provide a moderate improvement in enzyme stability. Stability of enzymes adsorbed onto nanoparticles is highly dependant on nanoparticle size and adsorption pattern. Protein and nanoparticle interactions during adsorption can cause conformational changes to an enzyme's native structure, rendering it inactive. Entrapment of enzymes has been demonstrated to improve the stability by restricting their ability to unfold. Entrapment of oxidase enzymes within inorganic silica nanogels were shown to improve the stability over the native form by up to 200-fold. To date, the optimal method for enhancing the stability of enzymes has been three-dimensional covalent immobilization of enzymes. LeJeune and Russell demonstrated that hydrolase enzymes which detoxify chemical warfare agents could be immobilized within polyurethane foams. The surface lysine residues participate in the crosslinking reaction by condensing with the isocyanate groups on the polyurethane backbone, resulting in a foam material that contains active enzymes which retain superior stability over the native enzyme [see, LeJeune, K. E., “Covalent binding of a nerve agent hydrolyzing enzyme within polyurethane foams”, Biotechnology and Bioengineering, Vol. 51, pages 450-457 (1996), and LeJeune, K. E., “Dramatically stabilized phosphotriesterase-polymers for nerve agent degradation”, Biotechnology and Bioengineering, Vol. 54, pages 105-114 (1997)]. This work has been extended to numerous enzymes which have been utilized to make colorimetric sensor pens that have shelf-lives of years and can withstand harsh environmental conditions such as heat and solvents (see also U.S. Pat. Nos. 6,291,200; 6,673,565; 6,762,213; and 6,759,220).
Recently, there has been a large focus on nanoparticle development in many fields including: optics and coatings, clinical diagnostics, drug-delivery, and also in the development of novel materials such as self-healing and highly-porous plastics. Stabilization of covalently-immobilized enzymes within porous, hydrophilic nanogels has been demonstrated by several groups. Polymers which respond to specific stimuli, such as temperature and the presence of other molecules in solution are frequently utilized in particle development. Nanoparticles composed of such polymers have the capability to shrink and swell via changes in Gibbs free energy in the presence of the proper stimulus. Responsive nanoparticles are currently used for drug delivery, bioimaging and therapeutics. The present invention provides three-dimensional immobilization of enzymes at the nanoscale within thermally responsive polymer materials which will protect the enzyme by providing a responsive barrier material that will respond to environmental stimuli to provide structural support under conditions that would otherwise denature the enzyme.
Enzymes have been functionalized and coupled with N-isopropylacrylamide (NiPAAm) with N-hydroxysuccinimide (NHS) [Chen, G., “Preparation and properties of thermoreversible, phase-separating enzymes-oligo (N-isopropylacrylamide) conjugates”, Bioconjugate Chemistry, Vol. 4, pages 509-514 (1993)]. NiPAAm is a thermo-responsive polymer that which undergoes a volume transition at temperatures above its lower critical solution temperature (LCST) approximately 32° C. Coupling NiPAAm to an enzyme allows it to be used for separation, recovery, and recycling of an enzyme simply by applying small temperature changes to the reaction medium. The growing NiPAAm enzyme chains have also shown moderate improvements in stability compared to native enzyme. However, heretofore, no one has cross-linked an enzyme or encapsulating an enzyme within thermally responsive (thermoresponsive) nanoparticles, as set forth in the present invention.
Incorporation of functional enzymes into nanoparticles is difficult for several reasons. Bottle-in approaches have limited utility because diffusion of enzymes into polymer particles, on a short time scale, is difficult due to small pore size and high polymer concentration on the outer particle shell. Harsh conditions during nanoparticle fabrication such as solvents, surfactants and high temperatures can be detrimental to the tertiary structure of the enzyme. The present invention provides an enzyme-friendly methodology for covalently immobilizing and encapsulating enzymes within stimulus responsive nanoparticles using standard oil-in-water emulsion polymerization protocols, such emulsion polymerization protocols are known by those persons skilled in the art. Essentially, hydrophobic graft-modified enzymes can be used as seeds in micelle systems for growth of nanoparticles. Incorporating functional enzymes into nanoparticles which are constructed from responsive polymers will further stabilize enzymes in harsh environments (for example, elevated temperature, chemicals, unfavorable pH, physical forces—all stressful).
Enzymes modified with NiPAAm polymers have shown an increased thermal stability over native enzymes. Through encapsulating and immobilizing an enzyme within responsive NiPAAm nanoparticles, the stability will be greatly improved at elevated temperatures; leading to a drastic improvement in both pot life (aqueous state) and shelf life (dry state) stability. The contraction of the particles supports the enzyme's tertiary structure, leaving the enzyme highly folded and active at elevated temperatures; free enzymes which are unencapsulated will unfold at these temperatures and become inactive.
The resulting functionalized enzyme conjugate-nanoparticle systems of the present invention have numerous applications. Enzymes generally demonstrate immense utility for a variety of industrial catalysis reactions; however the byproducts or intense environmental conditions limit the efficiency of using enzymes. The nanocatalysts and nanoparticles of the present invention stabilize enzymes to survive such intense environmental conditions including, such as for example but not limited to, temperature extremes. The nanocatalysts and nanoparticles of the present invention have high degree of utility for decontamination, chemical remediation, drug delivery, wound healing, protein therapy and a host of other applications.